Introduction
Environmental toxicity from non-essential heavy metals such as cadmium (Cd), which is released from human activities and other environmental causes, is rapidly increasing (Ahsan et al., 2009). In humans, Cd causes diseases such as osteoporosis and emphysema by damaging the lungs, kidneys, and bones (Kazantzis, 2004; Straif et al., 2009). In plants, Cd damages the photosynthetic apparatus, interrupts respiratory and nitrogen metabolism, and unbalances water and nutrient uptake (Herbette et al., 2006; Balen et al., 2011), ultimately reducing biomass, causing leaf chlorosis, inhibiting root growth, and even leading to plant death (Lin et al., 2007; Yadav, 2010; Lin and Arats, 2012). Additionally, plants can accumulate high Cd contents in their edible parts, which poses a potentially major hazard to human health (Satarug et al., 2003).
Zinc (Zn), an essential metal and a cofactor of numerous plant proteins and enzymes, plays several crucial roles in protein binding, enzyme activity, transcriptional and translational regulation, and signal transduction (Broadley et al., 2007; Lin and Arats, 2012). However, excess Zn can also cause toxicity, as it can damage DNA replication and disrupt enzyme activities and protein folding and function, ultimately inducing chlorosis and inhibiting plant growth and development (Broadley et al., 2007; Lin and Arats, 2012; Schneider et al., 2013).
Cd and Zn coexist naturally in the soil. Due to their physical and chemical similarities (Chesworth, 1991), their uptake and transport in plants use similar pathways (Grant et al., 1998). Many metal transporters that transport both Cd and Zn have been identified, including AtNRAMP3 and AtNRAMP4 (Thomine et al., 2000; Lanquar et al., 2010). In response to Cd and Zn stresses, plants have developed strategies to prevent Cd-induced damage and maintain Zn homeostasis. Therefore, researchers have investigated the various synergistic and/or antagonistic interactions of Cd/Zn and found these interactions to depend on species, external bioavailable metal concentration, tissue type, and developmental stage. For example, some durum and bread wheat show antagonistic interactions of Cd/Zn in which Cd uptake is inhibited by Zn and Zn uptake is inhibited by Cd in roots, stems, and leaves (Hart et al., 2002, 2005; Sun et al., 2005). Conversely, some wheat under field conditions has shown synergistic interactions in which Cd and Zn uptake are promoted by each other (Nan et al., 2002).
However, previous studies on Cd/Zn interactions mainly focused on their transport and biochemical responses (Hart et al., 2002, 2005; Nan et al., 2002; Hassan et al., 2005; Sun et al., 2005). Although proteomic changes in response to Cd or Zn have been successfully investigated using a proteomics approach (Kieffer et al., 2008, 2009; Ahsan et al., 2009; Fukao et al., 2011; Lin and Arats, 2012; Schneider et al., 2013), the molecular mechanisms of Cd/Zn interactions are unknown, which limits our understanding of the interactions of Cd/Zn. Polish wheat (2n = 4x = 28, AABB, Triticum polonicum L.), which has low genetic similarity with T. aestivum (Wang et al., 2013; Michalcová et al., 2014), accumulates high concentrations of Zn, Fe, and Cu and therefore has attracted the interest of producers and breeders (Wiwart et al., 2013). Meanwhile, dwarf polish wheat (DPW), collected from Tulufan, Xingjiang, China, shows high tolerance to Cd and Zn because its growth is not affected by the accumulation of high concentrations of these metals in seedlings (Wang X. et al., in press). However, molecular responses to Cd and Zn remain unknown. Since DPW accumulates high concentrations of Cd and Zn in seedlings, it is a useful system for studying Cd/Zn interactions.
The purposes of this study are to understand molecular responses to Cd and Zn stresses, to investigate Cd/Zn interactions in DPW seedlings and to understand the molecular mechanisms of Cd/Zn interactions in DPW roots using isobaric tags for relative and absolute quantification (iTRAQ). iTRAQ is a high-throughput proteomic technology (Karp et al., 2010) that has been successfully used to reveal plant responses to heavy metals (Ahsan et al., 2009; Fukao et al., 2011).
Discussion
Interactions between Cd and Zn have previously been shown to be antagonistic and/or synergistic in various plants (Hart et al., 2002, 2005; Sun et al., 2005; Tkalec et al., 2014). In the present study, Cd uptake was inhibited by Zn and Zn uptake was inhibited by Cd in DPW roots (Figure 1). Cd transport from root to shoot was inhibited by Zn (Figure 1A) but was promoted by Zn after 5 days after treatment (unpublished data). Meanwhile, Zn transport from root to shoot was not affected by Cd (Figure 1B). These results indicate that Cd and Zinc interact antagonistically in DPW seedlings, as previously reported in bread and durum wheat (Hart et al., 2002, 2005; Sun et al., 2005) and unlike the synergistic interactions that have been reported in other wheat under field conditions (Nan et al., 2002).
Proteomic changes in the roots implicated several proteins in the antagonistic interactions of Cd/Zn (Data Sheets 1–11). Two days after treatment, the relative abundances of 206 (Data Sheet 2), 303 (Data Sheet 3), and 190 (Data Sheet 4) proteins were induced in response to Cd, Zn, and Cd+Zn stresses, respectively (Figure 2). Among these, 53 proteins were induced in response to all three treatments, and 58, 131, and 47 proteins were induced in response to only Cd, Zn, or Cd+Zn stresses, respectively (Figure 2). We grouped these proteins into different interactions of Cd/Zn (Figure 2). Our results indicate that although Cd and Zn have similar physical and chemical properties (Chesworth, 1991) and pathways for uptake (Grant et al., 1998), they induce differential molecular responses (Lin and Arats, 2012), which result in the antagonistic interactions of Cd/Zn in DPW roots (Figure 1) and the high tolerance of DPW to Cd and Zn toxicity (Wang X. et al., in press). Some proteins identified in this study that are involved in noteworthy processes are discussed below.
To overcome oxidative toxicity caused by heavy metal stresses (Ranieri et al., 2005; Lin et al., 2007; Kieffer et al., 2008; Di Baccio et al., 2011; Zeng et al., 2011), plants utilize an effective antioxidant system that protects their cells against oxidative damage (Kieffer et al., 2008; Di Baccio et al., 2011) by inducing the expression of oxidation-reduction-related proteins (Lin et al., 2007; Kieffer et al., 2008, 2009; Di Baccio et al., 2011; Zeng et al., 2011; Schneider et al., 2013). In this study, 31 oxidation-reduction-related proteins were observed (Table 1). Of these, 8, 11, and 4 proteins were altered in response to Cd, Zn, and Cd+Zn stresses, respectively (Table 1). These results suggest that Cd, Zn, and Cd+Zn stresses cause differential oxidative threats which are detoxified through the induction of different oxidation-reduction-related proteins. Conversely, 8 Cd-induced proteins, 11 Zn-induced proteins, and 2 proteins induced by both Cd and Zn stresses were not induced in response to combined Cd+Zn stress, which suggests that the oxidative threats caused by Cd and Zn stresses are not the same as those caused by Cd+Zn stress. These results indicate that Cd and Zn detoxify each other in combined Cd+Zn stress, resulting in their uptakes being inhibited by each other. As described in previous studies (Kieffer et al., 2008, 2009; Schneider et al., 2013), Cd and Zn induced a greater abundance of some oxidative stress-related proteins but also induced a lower abundance of other oxidative stress-related proteins (Table 1). Among these, 3 proteins were induced by all 3 stresses (Cd, Zn, and Cd+Zn) (Table 1), including ascorbate peroxidase (protein 285), L-ascorbate peroxidase 2 (protein 573), and peroxisome type ascorbate peroxidase (protein 511), which are key peroxide detoxification enzymes (Raven et al., 2004). These results suggest that ascorbate mediates Cd- and Zn-induced oxidative stress in plants (Kieffer et al., 2008).
In response to Cd and Zn stresses, plants form heavy metal-glutathione (GSH) or metal-phytochelation (PC) compounds for metal detoxification (Seth et al., 2012; Jozefczak et al., 2015). GSH metabolism-related proteins, such as glutathione S-transferase (GST) and glutaredoxin (Grx), are differentially induced by Cd or Zn stress (Ahsan et al., 2009; Alvarez et al., 2009; Kieffer et al., 2009; Smiri et al., 2011; Zeng et al., 2011; Schneider et al., 2013). Meanwhile, GSTs translocate compounds of GSH-cytotoxic substrates into vacuoles for detoxification (Kumar et al., 2013). In this study, all three stresses (Cd, Zn, and Cd+Zn) induced GST (protein 405), Grx (protein 141), lactoyglutathione lyase (proteins 424), and 2 sulfate metabolism-related proteins [sulfurtransferase (protein 540) and ATP sulfurylase (protein 112)] (Table 1), suggesting that sulfate availability for the synthesis of metal chelations such as GSH (Speiser et al., 1992) determines Cd and Zn tolerance (Nocito et al., 2006; Alvarez et al., 2009). Additionally, 3 GSH metabolism-related proteins, including glutamine synthetase cytosolic isozyme 1-2 (protein 210), GST (protein 696) and lactoyglutathione lyase (proteins 135), were induced only in response to Cd stress (Table 1), suggesting that Cd is detoxified through sequestration of GSH-Cd compounds into vacuoles and subsequent reduction of oxidative stress (Seth et al., 2012; Jozefczak et al., 2015). However, these proteins were not induced in response to combined Cd+Zn stress, which partly illustrates Zn detoxification of Cd. Interestingly, 2 GSTs (proteins 79 and 731) and 2 Grxs (proteins 384 and 472) were induced in response to both Zn and Cd+Zn stresses (Table 1), similar to the results obtained for some GSTs induced by Zn stress in Noccaea caerulescens (Schneider et al., 2013). These results suggest that these proteins participate in the detoxification of Zn stress-induced reactive oxygen species (Dixon et al., 2009; Schneider et al., 2013).
As a precursor of GSH, SAM plays important roles in protecting against Cd stress-induced reactive oxygen species (ROS) (Noriega et al., 2007). In the present study, protein levels of serine hydroxymethyltransferase (SHMT) and spermidine synthase 1, key enzymes in SAM metabolism, were altered in response to both Cd and Zn stresses, suggesting that SAM plays important roles in protecting against these stresses. S-adenosylmethionine synthase (SAMS) synthesizes SAM, which is a precursor of nicotianamine (NA) (Schneider et al., 2013). The protein level of nicotianamine synthase 2 (NAS), which synthesizes nicotianamine (NA) from SAM, increased in response to Cd stress. NA is an essential compound for cell-to-cell transport of Zn, Fe, and Cu (Takahashi et al., 2003; Klatte et al., 2009). However, the abundances of both SAMS and NAS decreased in response to combined Cd+Zn stress, whereas a previous report in N. caerulescens showed increased SAMS and NAS levels in response to Zn stress (Schneider et al., 2013). Our results partially illustrate Cd inhibition of Zn uptake. Cd stress also causes the lignification of roots (Finger-Teixeira et al., 2010). SAM provides the methyl donor to caffeic acid 3-O-methyltransferase (COMT) in lignin biosynthesis (Wang Y. et al., 2016). COMT levels were increased only in response to Cd stress (Table 1), suggesting that Cd also causes root lignification.
Some organic acids such as oxalate, malate, citrate, and fumarate are induced by Cd and Zn stress (Ueno et al., 2005; López-Millán et al., 2009; Zhu et al., 2011; Schneider et al., 2013) and form metal-organic acid complexes to act as metal chelators to promote detoxification in planta (Verbruggen et al., 2009). Further, Cd and Zn also induce key enzymes that participate in organic acid metabolism (López-Millán et al., 2009; Schneider et al., 2013). In this study, Zn stress induced several of these enzymes (Table 1), including malate dehydrogenase (protein 58), isocitrate dehydrogenase (protein 225), aconitate hygratase (protein 39), citrate synthase 4 (protein 82), and oxalate oxidase GF-2.8 (protein 376). These results suggest that detoxification of Zn could be achieved through the formation of Zn-organic acid complexes and subsequently, the complexes are deposited into vacuoles (Schneider et al., 2013). Conversely, organic acid secretion is associated with Cd and Zn exclusion (Zhu et al., 2011). Combined Cd+Zn stress resulted in decreased abundances of furmarate hydratase 2 (protein 116), malate dehydrogenase 1 (protein 537) and succinate dehydrogenase (815), which are key enzymes in furmarate, malate, and citrate metabolisms, respectively (Table 1). Thus, our results partially illustrate the mutually inhibited uptake of Cd/Zn in the roots (Figure 1).
Cellulose and pectic polysaccharides are major components of the plant cell wall (Cosgrove, 2005), which can be modified by some heavy metals. For example, Cd enhances the contents of glucose and polysaccharides in cell walls (Li et al., 2015). Further, exogenous glucose alleviates Cd toxicity by increasing Cd fixation in root cell walls (Shi et al., 2015). In this study, several sucrose metabolism-related proteins were induced by Cd, Zn, or Cd+Zn stress (Table 1), suggesting that glucose and/or polysaccharides participate in Cd and Zn fixation, exclusion or sequestration in root cell walls (Li et al., 2015; Shi et al., 2015). However, 8 sucrose metabolism-related proteins were induced in response to Zn stress but not combined Cd+Zn stress (Table 1), which suggests that Cd detoxifies excess Zn by inhibiting its uptake, resulting in Cd-induced inhibition of Zn modification of sucrose metabolism.
Zn stress also affects the expression of P-type ATPases and several other metal transporters (Schneider et al., 2013). P-type ATPases, such as AtHMA4 from Arabidopsis, GmHMA3 from soybean and AhHMA3 from A. halleri, have Zn uptake activity (Becher et al., 2004; Hussain et al., 2004; Wang et al., 2012). Further, AtHMA2, 3, and 4 have been shown to transport Zn from root to shoot (Williams and Mills, 2005). In this study, the abundance of a P-type proton pump ATPase (protein 679) decreased in response to Zn stress but not combined Cd+Zn stress (Table 1), a result which contradicts previous work in N. caerulescens showing increased abundance of two P-type ATPases in response to Zn stress (Schneider et al., 2013). However, we found that other Zn-induced metal transporters were not observed 2 days after treatment, but their transcripts were regulated by Cd, Zn, and Cd+Zn 5 days after treatment (unpublished data).
Finally, as reported by previous studies (Di Baccio et al., 2011; Zeng et al., 2011), proteins that were similarly or differentially induced in response to Cd, Zn, and/or Cd+Zn stresses also participated in other processes, including ribosome biogenesis, DNA metabolism, protein folding/modification (all SFiles), suggesting that these proteins might contribute to differential defense mechanisms against these stresses (Zeng et al., 2011).